1. Field of the Invention
The present invention relates to the detection of antigens in a sample using antibody reagents.
2. Description of the Background
The evolving field of immunohistochemistry has led to simplified protocols with shorter incubation times, and automation. The ability to stain two or more antibodies on one tissue section has evolved as well. A double or triple stain assay would be much more practical for both pathologist and histo-technologist, reducing error, reducing reagents, reducing amount of tissue needed, and most important-time. Furthermore, this technology leads to a more practical method for a pathologist to make a diagnosis with antibodies that need to be stained in unison as well as situations where there is limit tissue.
In the past, double or triple stains were primarily performed in the research setting. However, the prior double and triple stained procedures required trained personnel, were technically difficult, time consuming, and in most cases, was not used on an automated immunostaining system. Therefore, the use of double or triple staining techniques is not often used in the clinical setting.
Previously, a typical 3-step avidin-biotin horseradish peroxidase (HRP) system was used for the first antibody sequence, followed by an elution (denaturing) step, and then a second antibody sequence with an alkaline phosphatase (AP) system. This product a brown (DAB) and red (Fast Red) color precipitate for each antigen, thus producing a double stain. If the tissue contained endogenous biotin, an avidin-biotin step was required. A double stain could require 30 to 40 steps and a triple stain could take 40 to 50 steps, depending on the complexity of the assay. Most automated immunohistochemistry stainers were not designed to do these complicated double stain assays. It also required a highly trained technician to properly titer each antibody sequence.
A biotin-free polymer detection kits for both HRP and AP detection system has been recently introduced (DakoCytomation; Zymed Laboratories). These kits eliminate the avidin-biotin blocking steps and require one step for detection versus two. However, depending on the immunostainer and the number of washes required, these kits still require significant technical expertise and IHC staining programming are not design adequately for double stains and especially triple stains. Also, lack of sensitivity with nuclear antigens have been reported with this kind of polymer kit. This new technology has been popular in the research arena, but has not been used much in clinical setting because of these difficulties.
The use of antibody cocktails has been used in the clinical setting for a number of years. LCA, AE1/AE3, CMV, and most recently, CD15, Pan Melanoma (HMB45+MART-1+Tyrosinase) and PIN-4 (P504S+HMWCK+p63) are several cocktails that are being used on a routine basis in the clinical setting. Universal detection kits with both anti-mouse and anti-rabbit have also been used since the late 1980's.
Double and triple stain technology using immunohistochemistry in formalin-fixed paraffin-embedded tissues has also been used for many years. Double stains are accomplished by applying primary antibodies and detection in a sequence of steps to achieve multiple labeling on the same tissue. Various methods for detection used include fluorescence, immunoperoxidase, immunogold and in situ methods.
In view of the above, the disadvantages of the previous double stain technology have become apparent. Illustrative of these disadvantages is the typical procedure of the previous technology to perform a double stain immunoassay. For example, the sample is treated with Hydrogen Peroxide for 5 minutes followed by two optional protein block (5 to 10 minutes) and Avidin-Biotin block (20 to 40 minutes). Then, the primary antibody is applied for 30 to 60 minutes, linked for 10 to 20 minutes, labeled with, e.g., HRP for 10 to 20 minutes, treated with DAB for 5 minutes, and then denatured for 5 minutes. Subsequently, the second primary antibody is applied for 30 to 60 minutes, followed by an optional protein block for 5 to 10 minutes, linking for 10 to 20 minutes, and then labeling the second primary antibody, for example, with AP, for 10 to 20 minutes. The reactivity is detected by applying Fast Red for 10 to 20 minutes followed by counterstaining (plus bluing) for 30 and 60 seconds and coverslipping, which requires a water-base mounting media. The total time for this double stain procedure is about 3 to 4 hours with a total of 11-15 manual steps; plus 12 to 14 washes (32 maximum steps). Further, for a triple stain one would add 5 more steps (40+ maximum steps) and would take a total time of 4 to 5 hours.
Generally, diluting antibodies in a buffer and storing antibodies is known however, those dilutions typically were performed in phosphate buffered saline or other isotonic solution and may also have small quantities of bovine serum albumin and in some instances relatively small amounts of detergents such as Triton X-100 or NP-40 (Antibodies: A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; and Current Protocols in Molecular Biology, Ausebel et al (eds.), John Wiley and Sons, Inc. N.Y., 2001). However, the combination of reagents in a primary antibody cocktail as described as the present invention was not described previously. Furthermore, it was not known that these cocktails would stabilize the antibodies and permit the rapid and efficient staining with two or more antibodies.